Use of shroom3 in chronic kidney disease and chronic allograft nephropathy

ABSTRACT

A method for identifying the risk of developing Chronic Allograft Nephropathy (CAN) in a patient that received a kidney transplant from a donor which comprises identifying the race of the donor; determining the levels of SHROOM 3 expression in a kidney biopsy specimen obtained from the patient at a predetermined time after transplant; comparing the level of SHROOM 3 expression in the biopsy specimen with the levels of SHROOM 3 expression in a control; determining if the level of SHROOM 3 expression in the allograft is significantly higher than in the control, and diagnosing the patient as being at risk for CAN if the level of SHROOM 3 expression in the specimen is significantly higher than in the control.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional, and claims priority to U.S.application Ser. No. 14/773,687, filed Sep. 8, 2015, now U.S. Pat. No.11,098,361, issued Aug. 24, 2021, which is the U.S. National PhaseApplication under 35 U.S.C. § 371 of International Patent ApplicationNo. PCT/US2014/022607 filed Mar. 10, 2014, which claims the benefit ofU.S. Provisional Application No. 61/777,328 filed Mar. 21, 2013, all ofwhich are incorporated by reference herein. The InternationalApplication was published in English on Oct. 2, 2014 as WO2014/159227 A1under PCT Article 21(3).

GOVERNMENT CLAUSE

This invention was made with government support under 1U01AI070107-01awarded by The National Institutes of Health. The government has certainrights in the invention.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submittedelectronically as an ASCII text file named 27527-0124002SEQ.txt. TheASCII text file, created on Sep. 21, 2021, is (8,192 bytes in size. Thematerial in the ASCII text file is hereby incorporated by reference inits entirety.

FIELD OF THE INVENTION

The present invention pertains to methods to identify patients sufferingfrom kidney diseases and predict the development and progression ofkidney fibrosis in renal allograft recipients. The invention includes akit for identifying patients suffering from kidney diseases and forpredicting the development and progression of kidney diseases andtubulo-interstitial fibrosis in renal allograft recipients.

BACKGROUND OF THE INVENTION

Chronic Kidney disease (CKD) affects 10% of US adults with risingincidence and prevalence worldwide {Coresh J, 2007}. End-stage renaldisease (ESRD) requires renal replacement therapy (RRT) and currentlyaffects over 500,000 US adults. In addition to conferring risk for endstage renal disease (ESRD), CKD increases the risk of cardiovasculardisease and all-cause mortality {Weiner D, 2004}. Tubulo-interstitialfibrosis (TIF) is a final common pathogenic process for CKD from variedetiologies leading to the development of ESRD. TIF is also a primarycomponent of chronic allograft nephropathy (CAN) and is associated withprogressive decline of estimated glomerular filtration rate (eGFR) inthe renal allograft {Chapman J, 2005} {Nankivell B J, 2003}. CANrepresents the most common cause of death-censored long-term graft lossand is measured histologically by the chronic allograft dysfunctionindex score (CADI score) {Isoniemi H, 1992} {Yilmaz S, 2007}. To date,there is no effective anti-fibrotic therapy to prevent the progressionof CKD or CAN. Presently, some patients with CKD or CAN will eventuallyprogress to ESRD and need RRT. For instance, renal allograft recipientsrepresented 13.5% of patients on the kidney transplant waitlist and 15%of all transplants performed in 2005 {Magee J C, 2007}. Renal allograftrecipients have a 30% probability of requiring RRT or re-transplantationat 10-years {USRDS 2012}. RRT and ESRD-care are a disproportionate andburgeoning financial burden to Medicare {Iglehart, 2011}. It istherefore critical to identify new sensitive biomarkers to predict thedevelopment of kidney fibrosis. Furthermore, these markers couldrepresent targets for therapeutic intervention to prevent thedevelopment of TIF at an early stage, thereby preventing progression toESRD.

Allograft biopsy based studies (both for-cause and protocol) haveprovided insight into how early allograft changes correlate with longterm allograft outcomes {Gago M, 2012} {Rush D, 1994} {Seron D, 1997}{Cosio F, 2005} {Park W, 2010}. More recently, distinct biopsy and bloodgene-expression profile (transcriptome) signatures have been shown toclassify patients with acute rejection, chronic rejection, those onimmunosuppression, and operationally tolerant recipients {Akalin E,2010} {Akalin E, 2001} {Flechner S, 2004} {Donauer J, 2003} {Sarwal M,2003} {Reeve J, 2009} {Scherer A, 2003} {Sagoo P, 2010} {Newell K,2010}. These gene panels have been able to improve upon histologicalclassifiers alone {Sarwal M, 2003} {Reeve J, 2009}. Genome-wideassociation studies have also strongly linked a single-nucleotidepolymorphism (SNP) in the Shroom3 gene (rs17319721) with incident andprevalent CKD by eGFR in population-based cohorts of European ancestry{Kottgen A, 2009} {Boger C, 2011}. The Shroom3 gene encodes a PDZdomain-containing protein that can directly bind F-actin and regulateits subcellular distribution in cells. Complete absence of or defectiveShroom3 causes open neural tube defects and neonatal death in mice{Hildebrand J, 1999}. In MDCK kidney cell lines, Shroom3 localizes atthe apical and junctional complexes and is critical to the maintenanceof normal epithelial cell phenotype {Hildebrand J, 2005}. It is alsoknown that C-terminal domain of Shroom3 interacts with Rho-Kinases(ROCKs) to facilitate myosin phosphorylation and actin contraction{Nishimura T, 2008}. However, whether Shroom3 plays a role in kidneyfibrosis in CKD or CAN is yet unknown.

What are needed in the art are markers whose expression can be used toidentify patients suffering from kidney diseases and predict thedevelopment of kidney fibrosis. In addition, such markers are needed toidentify renal allograft recipients who are at risk for developing CANand represent targets for therapeutic intervention to prevent thedevelopment of TIF at an early stage, thereby preventing progression toESRD.

SUMMARY OF THE INVENTION

Shroom3 is a novel candidate gene whose expression in a renal allograftprecedes and predicts decreased renal function and TIF. Higher allograftShroom3 levels predict histological progression of CAN. It has also beenfound that these relationships correlate best in recipients ofwhite-donor kidneys. The findings confirm for the first time that thepreviously described chronic kidney disease (CKD)-associated Shroom3locus (rs17319721) {Kottgen A, 2009} {Boger C, 2011} mediates its effectthrough increased Shroom3 expression. In addition, it has beendiscovered that Shroom3 has a salutary role in canonical TGF-betasignaling and Collagen-1 production.

In one aspect, the present invention provides method for identifying therisk of developing Chronic Allograft Nephropathy (CAN) in a patient thatreceived a kidney transplant from a donor which comprises identifyingthe race of the kidney donor; determining the levels of SHROOM3expression in a kidney biopsy specimen obtained from the patient at apredetermined time after transplant; comparing the level of SHROOM3expression in the biopsy specimen with the levels of SHROOM expressionin a control; determining if the level of SHROOM3 expression in theallograft is significantly higher than in the control, and diagnosingthe patient as being at risk for CAN if the level of SHROOM3 expressionin the specimen is significantly higher than in the control.

In a further aspect the present invention provides a method foridentifying the risk of developing Chronic Allograft Nephropathy (CAN)in a patient that received a kidney from a donor comprising the steps ofidentifying the race of the kidney donor; obtaining a renal allograftbiopsy sample from the patient; determining the levels of SHROOM3expression in said biopsy; comparing the levels of SHROOM3 expression insaid biopsy with the levels of SHROOM3 expression in a control andadvising the patient as being at risk of developing CAN if the levels ofSHROOM3 in the sample are significantly higher than in the control andthe kidney donor is Caucasian.

In yet a further aspect, the present invention provides a method foridentifying the risk of developing a renal disease in a Caucasianpatient that received a kidney from a donor, comprising the steps ofdetermining if said donor expresses the rs 17319721 SNP risk allele andconducting a genetic analysis to determine if said donor is homozygousfor said risk allele, wherein if said donor is homozygous for said riskallele then said patient is at risk for developing a renal disease.

In yet a still further aspect, the present invention provides a methodfor identifying the risk of developing fibrosis in a Caucasian kidneydonor, comprising the steps of determining if said donor expresses thers 17319721 SNP risk allele, and conducting a genetic analysis todetermine if said donor is homozygous for said risk allele; wherein ifsaid donor is homozygous for said risk allele then said donor is at riskfor fibrosis.

In a still further aspect the present invention provides a method foridentifying the risk of developing a progressive kidney disease selectedfrom the group consisting of Chronic Allograft Nephropathy (CAN) andChronic Kidney Disease (CKD) of a Caucasian patient which comprises:determining the levels of SHROOM3 expression in a kidney biopsy specimenobtained from the patient at a predetermined time, comparing the levelof SHROOM3 expression in the biopsy specimen with the levels of SHROOM3expression in a control, determining if the level of SHROOM3 expressionin the specimen is significantly higher than in the control, anddiagnosing the patient as being at risk for said disease if the level ofSHROOM3 expression in the specimen is significantly higher than in thecontrol.

In a still further aspect, the present invention provides a kit foridentifying patients suffering from a renal disease and at risk fordeveloping CAN or CKD comprising in separate containers primers for usein RT-PCR assays for SHROOM3 expression, RT-PCR for detecting the rs17319721 SNP risk allele, a positive control, buffers and instructionsfor use.

These and other aspects of the present invention will be apparent tothose of ordinary skill in the art in light of the present description,claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: a diagram depicting the number of participants in the studyreported herein, and showing that 160 participants had 3-month allograftbiopsy RNA extracted for microarray. Among these 160 patients, at12-months post-transplant, 147 had eGFR-creatinine, 101 had CADI-12 and85 had CADI-3 and -12 reported.

FIG. 2A-2D: graphs depicting (2A) the correlation of allograft logShroom3 expression (microarray) at 3 months and eGFR creatinine at 12months (r=−0.20; p=0.007); (2B) the correlation of allograft log Shroom3expression (microarray) at 3 months and CADI-12 months (Pearson R-0.22;p=0.027; (2C) Shroom3 expression was higher in all donors withprogressive fibrosis (Delta CADI=2 or more; n=17) vs those withoutsignificant progression (Delta CADI<2; n=68); and (2D) the correlationbetween 3-month Shroom3 expression was strongest in deceased-donorkidneys and 12-month CADI (r=0.34; p=0.0088) [Line represents mean;Whiskers=SD].

FIG. 3A-3D: graphs depicting (3A) the correlation of Fold change Shroom3expression (RT-PCR) at 3 months and CADI-12 months (Pearson r=0.4169;p=0.0103); (3B) the correlation of Fold change Shroom3 expression(RT-PCR) at 3 months and eGFR creatinine at 12 months (r=0.3230; p=(3C)the correlation between allograft log Shroom3 expression (microarray) at3 months and Fold change Shroom3 expression at 3 months (RT PCR)(Pearson r=0.5613; p=0.0008); and (3D) the regression line ofrelationship between log Shroom3 expression at 3 months and simultaneousCADI score—No relationship could be identified

FIG. 4A-4C: graphs depicting (4A) the correlation of allograft logShroom3 expression at 3 months and CADI-12 months in recipients ofwhite-donor kidneys (r=0.2538; p=0.02); (4B) the correlation ofallograft log Shroom3 expression to eGFR-creatinine in recipients ofwhite-donor kidneys (r=−0.25; p=0.008); and (4C) Log Shroom3 expressionwas higher in WDKRs with progression of fibrosis (Delta CADI≥2; n=14) vsthan those without significant progression (Delta CADI<2; n=56)[*p<0.05; **p<0.001; ***p<0.0001]

FIG. 5A-5C: graphs depicting that (5A) there is no correlation betweenallograft log Shroom3 expression (microarray) at 3 months and 12-monthCADI in living donor recipients (LDRs) and: (5B) in non-WDKRs; (5C) Nocorrelation between 3-month Shroom3 and eGFR-12 m in non-WDKRs.

FIG. 6A-6B: graphs illustrating that (6A) Shroom3 SNP (rs17319721) isdifferently distributed between whites and non-whites (amongst bothdonors and recipients). Whites have a higher prevalence of effect allele(p<0.0001) (6B): Homozygosity for the risk allele in donor kidneys isassociated with significantly increased allograft Shroom3 expression(p=0.033) whiskers: 5th-95th percentile; Line at Median; p=0.0183)[*p<0.05; **p<0.001; ***p<0.0001]

FIG. 7A-7C: graphs depicting that (7A, 7B, respectively) Shroom3expression was increased in both WDKRs and non-WDKRs with the presenceof effect-allele (A) in the donor and (7C). Shroom3 expression was notsignificantly affected Recipient SNP type (whiskers: Min-Max; Line atMedian; p=0.0309) [*p<0.05; **p<0.001; ***p<0.0001]

FIG. 8A-8B: graphs that illustrate (8A) Allele prevalence of the Shroom3SNP in 354 white recipients (as %) according to ESRD etiology.Recipients with ESRD from Diabetes (49%) had the significantly greaterrisk-allele prevalence compared to unrelated donors. (8B): Alleleprevalence of the Shroom3 SNP in 3247 patients of the study. Risk alleleprevalence was highest in Diabetics with CKD (51%) [*p<0.05; **p<0.001;***p<0.0001].

FIG. 9: a construct map of Luciferase-reporter plasmids used in Example5 below.

FIG. 10A-10B: FIG. 10A is a graph that depicts that Luciferase-reporterplasmids with A-allele enhancer element showed greater activity thanG-allele and promoter-only plasmids; FIG. 10B is a Western Blot thatillustrates that Nucleoprotein extracted from 293-T cells showedenhanced binding to oligonucleotide sequences containing the G-allele.

FIG. 11A-11B: FIG. 11A is a graph illustrating that in PRCEC, TGF-βtreatment increases Shroom3 in a dose-dependent (up to 5 ng/ml) and timedependent fashion by RT-PCR (error bars: mean in PRCEC, TGF-β treatmentincreases Shroom3 in a time dependent fashion (up to 5 ng/ml) (errorbars: mean±SEM) [*p<0.05; **p<0.001; ***p<0.0001]. FIG. 11B is a WesternBlot that illustrates that TGF-β treatment increases Shroom3 in atime-dependent fashion.

FIG. 12A-12B: FIG. 12A is a graph that depicts the results of Example 6that shows TGF-beta increases Shroom3 expression in abeta-Catenin/TCF7l2 dependent manner. Quercetin (beta-Catenin inhibitor)and BC-21 (TCF7L2 inhibitor) inhibited the increase in Shroom3expression induced by TGF-beta (RT-PCR). 12B is a Western blot thatdepictsShroom3 protein increases in a Beta-Catenin/TGF-β dependentmanner.

FIG. 13A-13B: FIG. 13A is a chart depicting Shroom3 overexpression inPRCEC with PC-SHROOM3 transfection confirmed by RT-PCR and FIG. 13B byWestern blot respectively (mean±SEM) [*p<0.05; **p<0.001; ***p<0.0001.

FIG. 14A-14C: FIG. 14A is a graph depicting the results of Example 7 andshowing the effect on TGF-beta induced expression of profibrotic matrixmarkers by RT-PCR. Shroom3 over-expression alone increased Collagen-1and Fibronectin production while knockdown suppressed these matrixmarkers (mean±SEM) [*p<0.05; **p<0.001; ***p<0.0001]; FIG. 14B is aWestern blot showing Shroom3 over-expression enhanced Phosphorylation ofSmad-3 in response to TGF-beta in PRCEC at 15 minutes (Right); FIG. 14Cis a photograph showing that TGF-β1 treatment of SHROOM3-overexpressedPRCEC also developed more prominent F-actin bundles compared toTGFβ1-treated cells without SHROOM3 overexpression (FIG. 14C—Upper andlower Panels).

FIG. 15A-15B: Graphs that depict the results of Example 7 whichestablishes that Shroom3 overexpresses in PRCEC with PC-SHROOM3transfection, confirmed by RT-PCR (FIG. 15A) and Western blot (FIG. 15B)respectively (mean±SEM) [*p<0.05; **p<0.001; ***p<0.0001]

FIG. 16A-16D: (16A) Representative genotyping results for ROSA-RTTA(upper) and COLTGM (lower) are depicted. (16B) Western blots ofphospho-SMAD3 (p-SMAD3), total SMAD3 (SMAD3), Shroom3 and β-actin frommouse kidney cortex lysates from UUO kidneys of non-Dox-fed vs Dox-fedmice are displayed (n=2). (16C) Left to right—Photographs showingrepresentative, low power (10×) images from Control kidney and UUOKidney of non-Dox-fed animals, and UUO kidneys of Dox-fed animals aredisplayed. Upper row—Periodic acid Schiff stain, middle row—Picrosiriusred stain. Lower row—immunolabeling for COL1A1 (TRITC-Alexa fluor) wasperformed on snap frozen kidney cortex sections. Representative imagesare displayed. Graph represents morphometric quantification of AreaCOL1A1 staining/total area (%) in 5 random high power fields per animal.(16D) Bar graphs depicting SHROOM3 and COL1A1 mRNA expression by RT PCRin control and UUO kidneys of DOX- and non-DOX fed animals (normalizedto GAPDH; n=5; mean±SEM; ANOVA with post-test Bonferroni comparison;*P<0.05).

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “about” or “approximately” usually means within an acceptableerror range for the type of value and method of measurement. Forexample, it can mean within 20%, more preferably within 10%, and mostpreferably still within 5% of a given value or range. Alternatively,especially in biological systems, the term “about” means within about alog (i.e., an order of magnitude) preferably within a factor of two of agiven value.

The term “significantly higher levels of Shroom3 expression” is definedherein as between about 1.4 and about 5-fold higher than in the control.

The present invention is based on the discovery that the levels ofSHROOM3 expression are significantly higher in kidney allograftrecipients that are at risk for developing CAN when compared to controlbiopsies obtained normal subjects. Pursuant to the present invention,SHROOM3 expression levels are determined from biopsies obtained fromkidney allograft recipients and compared to biopsies obtained fromcontrols which are normal kidney samples such as living donor baselinebiopsy samples or kidney samples from nephrectomy surgeries usingtechniques well known in the art such as immunostaining but preferablyby Real Time Polymerase Chain Reaction (RT-PCR).

In a Genomics of Chronic allograft rejection study protocol, biopsieswere obtained from all enrolled patients at different time points (SeeExamples below). From the DNA-microarray performed on allograft biopsiesat 3-months, genes whose differential expression correlated withCADI-score and renal function at 12-months were identified and ranked.Among the top ranked genes in this list was SHROOM3 (Unpublished data).

Genome-wide association studies have also strongly linked asingle-nucleotide polymorphism (SNP) in the SHROOM3 gene (rs17319721)with incident and prevalent CKD by eGFR in population-based cohorts ofEuropean ancestry {Kottgen A, 2009} {Boger C, 2011}. The SHROOM3 geneencodes a PDZ domain-containing protein that can directly bind F-actinand regulate its subcellular distribution in cells. Complete absence of,or defective SHROOM3 causes open neural tube defects and neonatal deathin mice {Hildebrand J, 1999}. In MDCK kidney cell lines, SHROOM3localizes at the apical and junctional complexes and is critical to themaintenance of normal epithelial cell phenotype {Hildebrand J, 2005}. Itis also known that the C-terminal domain of SHROOM3 interacts withRho-Kinases (ROCKs) to facilitate myosin phosphorylation and actincontraction {Nishimura T, 2008}.

In the study reported herein, 589 patients were enrolled. Allograftbiopsies were obtained at 0, 3, 12, and 24 months post-transplant withChronic allograft dysfunction index score (CADI) reported from a corelab. Gene expression microarray analysis was performed on 3 monthbiopsies (Affymetrix: human exon-1 chip) and correlation to 12-monthCADI and eGFR were analyzed (n=160). Overexpression and lentiviralsuppression studies were performed on human primary tubular cells(RPTE). SHROOM3 gene-expression was found to correlate linearly withfibrosis and negatively with eGFR at 1 year (n=101; p<0.05). This wasconfirmed by RT-PCR independently (n=36). No correlation was seenbetween SHROOM3 expression and 3 month CADI (n=137).

A SNP in SHROOM3 (rs17319721) has been linked to CKD in genome-wideassociation studies. As disclosed herein, it has been found that thepresence of at least 1 copy of the risk allele (i. e. A/G or A/A) in adonor's DNA is associated with higher intragraft SHROOM3 expression at 3months (n=136; p=0.02). The risk allele was more prevalent in white vs.non-white donors. As shown in Example 4 below, the SNP risk allele ismore frequent in white diabetics with renal disease. Therefore, thepresent invention provides a method for identifying the risk ofdeveloping CAN in a diabetic Caucasian patient suffering from renaldisease that received a kidney from a donor, comprising the steps ofobtaining a blood sample from the donor at baseline and conducting agenetic analysis to determine if the donor is homozygous for the riskallele. As shown in Example 9 below, the method can be practiced withnon-diabetic Caucasian patients. If the donor is homozygous for the riskallele then the patient is at risk for CKD or CAN. This method can onlybe performed if the donor is available, which is not always the case. Ifthe donor is not available then SHROOM3 expression levels in the patientcan be examined. While in recipients of white-donor kidneys SHROOM3expression was predictive of CADI at 12 months, this was not true fornon-white or living donor recipients.

Overexpression of SHROOM3 in RPTE increased, while lentiviralsuppression markedly diminished type-1 collagen production (p<0.01).SHROOM3 excess facilitated, while suppression inhibited canonicalTGF-beta signaling, evidenced by Phospho-Smad3 and profibrotic markerproduction. Further, in FVB/N mice with CKD (HIV-nephropathy andUnilateral Ureteric Obstruction), SHROOM3 expression was increasedcompared to controls as determined by RT-PCR. Therefore, the presentinvention is not limited to CAN but is useful for other CKD such asobstructive uropathy, HIV-associated nephropathy and diabeticnephropathy.

Without wishing to be bound by theory, it is believed that Shroom3 isinvolved in any disease that involves fibrosis such as liver fibrosisand lung fibrosis. Therefore, the materials and methods described hereinwill be useful for monitoring the progression of such diseases. As shownbelow in Example 10 renal interstitial fibrosis was significantlyabrogated in Shroom3 knockdown animals in a mouse model. These resultsvalidate the role of SHROOM3 in these diseases.

The present invention also provides kits for use in the methodsdisclosed herein. The kits comprise, in separate containers, thefollowing components: primers for RT-PCR SHROOM3 expression assays, amicroarray for gene expression analysis and primers for RT-PCR analysisof the rs17319721 risk allele, buffers and instructions for use. Anon-limiting list of primers for use in the Kits is set forth in Table5. The positive control comprises the cells over-expressing SHROOM3 orbrain tissues, such as neuroepithelium, which are known to have highlevels of SHROOM3 expression. (Shroom3-mediated recruitment of Rhokinases to the apical cell junctions regulates epithelial andneuroepithelial planar remodeling—Tamako Nishimura and MasatoshiTakeichi, Development 135, 1493-1502 (2008) doi:10.1242/dev.019646).

The present invention is directed to methods for identifying kidneyallograft recipients who are at risk for developing kidney diseases suchas CAN or CKD. When such patients are identified, the present inventionincludes methods for treating such patients. Such methods include,without limitation, administration of immunosuppressive drugs, i.e. acalcineurin inhibitor (CNI), such as cyclosporine or tacrolimus, or aless fibrogenic immunosuppressive drug such as mycophenolate mofetil(MMF) or sirolimus. Since patients who are identified as being at riskfor developing CAN or CKD have impaired renal function and often sufferfrom hypertension, administration of an angiotensin converting enzymeinhibitor (ACEI) such as lisinopril or angiotensin II receptor blockadesuch as losartan, to such patients is within the scope of the presentinvention.

The present invention also provides methods using SHROOM3 as a target toscreen for drugs useful for the treatment of CAN and CKD. The 293T cellstransfected with the SHROOM3 A-allele/luciferase construct described inExample 5 below can be used in screening assays to identify drugs forthe treatment of fibrotic diseases mediated by SHROOM3. The cells can beseeded in 96 well microplates, contacted with drug candidates andassayed for changes in luciferase activity. SHROOM3 activity (i.e.,luciferase) at baseline will be measured, and inhibitors Quercetinand/or BC-21, can be used as positive controls.

As an alternative, the 293T cells will be treated with TGFB1 (5-10ng/ml), a known up-regulator of SHROOM3 expression and these cells (witha higher baseline of SHROOM3 expression) are used in the screeningassays. These assays can be developed in a high throughput format.

Primers for use in RT-PCR assays for the A allele and for generatingluciferase reporter constructs are set forth in Table 5.

In summary, pursuant to the present invention, SHROOM3 has beenidentified as a novel candidate gene whose expression in the renalallograft precedes and predicts the derangement of renal function andTIF in CAN. Significantly higher allograft SHROOM3 levels can be used topredict histological progression of CAN. In addition, it has beendiscovered that these relationships are most predictive in recipients ofwhite-donor kidneys. These findings confirm for the first time that thepreviously described CKD-associated SHROOM3 locus (rs17319721) {KottgenA, 2009} {Boger C, 2011} mediates its effect through increased SHROOM3expression. Finally, the in vitro studies described herein suggest asalutary role for SHROOM3 in canonical TGF-beta signaling and Collagen-1production in renal tubular cells. Taken together, these findingsdemonstrate that SHROOM3 is a therapeutic target in both CAN and CKD andsuppression of its level can be used to inhibit the progression of TIFand retard ESRD.

The present invention is described below in working examples which areintended to further describe the invention without limiting the scopethereof.

In the Examples below, the following Materials and Methods were used.

Biopsies and RNA extraction: Real-time, ultrasound-guided, renalallograft biopsies were obtained at 0, 3, 12, and 24 monthspost-transplant, at 3 of 5 clinical sites. Two Cores were extractedusing 18 G spring-loaded biopsy needles when possible. If a single corewas obtained, preference was given to RNA extraction (QIAGEN-ALLprepkit, Valencia, Calif. USA) at the 3-month visit and to histologicalanalysis at 12-months. Tissue for gene-expression studies was storedimmediately in RNA-later and shipped at −20° C. to a genomics corefacility.

Reverse transcription: Extracted biopsy RNA were reverse transcribedusing Sensiscript single-step RT (Qiagen) and Oligo-DT primer (Qiagen)with starting total-RNA amount of 55 ng. Extraction samples with RNAconcentrations <5 ng/mcl by nanodrop were not used. For in vitro studieswe used Superscript-III (Invitrogen-Life technologies, Grand Island,N.Y.) with starting total RNA 500-1000 ng.

RT-PCR: Intron-spanning primer sets were designed for Shroom3 usingPrimer-BLAST (NCBI) and PCR amplicons were confirmed by both meltingcurve analysis and agarose gel electrophoresis. Shroom3 expression wasassayed in an internal and external cohort of patients by real-timepolymerase chain reaction (RT-PCR) (Applied biosystems 7500 cycler).

Shroom3 SNP analysis: Targeted genotyping was performed for Shroom3 SNP(rs17319721) using Taqman SNP analysis assay (Cat No: 4351379 AppliedBiosystems, Foster City, Calif.). DNA was extracted (QIAGEN-ALLprep kit,Valencia, Calif. USA) from pre-implantation biopsies or blood for donorSNP and from peripheral blood for recipient SNP assay. Automatedanalysis using Genotyping software from Applied Biosystems was performed(Applied Biosystems 7500 cycler).

Shroom3 promoter-enhancer constructs and Luciferase-reporter assay:Promoter fragments spanning −3000 bases 5′ of the translation start siteof SHROOM3 were PCR amplified using primer sets that optionally includedtwenty-four base-pair sequences of the intron-1 of SHROOM3 including thers17319721 site containing either of the two alleles (A or 1. G)(Table-1). Restriction sites for KpnI were introduced in all forwardprimer and Hind III in the reverse ones. The PCR products were thencloned into luciferase reporter vector-pGL3 Basic (E-1751, Promega, WI,USA) using Kpn I and Hind III sites. This generated 3 reporterplasmids—Promoter only, Promoter with either intronic-A or -G sequences.

Transient transfection of these constructs (1 mcg each) with Renillaluciferase reporter plasmid-pTL-TK (200 ng) was carried out inHEK293T-cells plated on 6-well plates at 80% confluence (Polyjetreagent, SignaGen labs, Rockville, Md.). After 24-hours, cells werelysed and protein extracted. Renilla and Luciferase activity wasmeasured in lysates using dual luciferase assay system (PJK Germany) onmicroplate reader according to manufacturer protocol. Results wereexpressed and Luciferase: Renilla ratio.

Western Blotting: Cells were lysed with a buffer containing 1% Triton, aprotease inhibitor mixture and tyrosine and serine/threoninephosphorylation and phosphatase inhibitors. Lysates were subjected toimmunoblot analysis using Rabbit anti-Shroom3 (a gift from Dr JeffreyHildebrand, Pittsburgh), anti-V5 tag antibody (R960-25, Invitrogen-Lifetechnologies), Phospho-Smad3 antibody (Rabbit polyclonal-pS423/425) andSmad-3 (Rabbit monoclonal Smad-3, #9523, from Epitomics, Burlingame,Calif.). Densitometry was performed as previously described {Gassman2009}.

Overexpression studies: A human Shroom3 construct (Open Biosystems,Lafayette, Colo.) was cloned into mammalian expression vector PC-DEST40(Invitrogen, Carlsbad, Calif.) with C-terminal V5 and Histidine tagsusing Clonase-II recombinase (Invitrogen). Electroporative transfectionusing Lonza Nucleofector Technology (Basic Nucleofector kit for PrimaryMammalian Epithelial Cells, Program T20) was used to transfect primaryrenal cortical epithelial cells (PRCEC) as described previously {Jin Y,2012}. Forced expression was confirmed in PRCEC by RT-PCR and Westernblot with empty destination vector transfection as control. Profibroticextracellular matrix markers were analyzed in PRCEC by Real-timepolymerase chain reaction (RT-PCR). Effect of TGF-β treatment on matrixmarker production in Shroom3-transfected cells was assayed in nutrientstarved medium 36 hours after transfection. Phosphorylation of Smad-3was measured at 5, 15 and 30 minutes of treatment with TGF-Beta.

Shroom3 SIRNA suppression studies: Human Shroom3 short hairpin clones(Open Biosystems, Lafayette, Colo.) were tested for Shroom3 suppressionby RT-PCR and Western blot in 293-T cells. The selected GFP-taggedhairpin was transfected into 293-T cells along with envelope plasmids(Polyjet reagent) to generate a mammalian VSV pseudotyped lentiviralexpression construct. PRCEC were infected using this lentiviralconstruct and Shroom3 suppression was confirmed. For in vivo Shroom3suppression in mouse, potent suppressive hairpin sequences wereshortlisted using a sensor Ping-Pong assay capable of decipheringelaborate shRNA libraries (Mirimus Inc., Long Island, N.Y.). Mousepodocytes were transduced with these sequences on a Mir30 lentiviralbackbone and tested for Shroom3 suppression using RT-PCR and Westernblot. Two sequences were selected for embryonic stem cell injection intomice to develop a Tetracycline-inducible Shroom3-ShRNA mouse model.

Characteristics of the Participants

Five hundred eighty nine recipients were enrolled in the study from 5centers at the completion of enrollment for the study. The demographicand clinical characteristics of donors and recipients in the cohort arelisted in Table-1. Demographics and clinical characteristics of patientsincluded in microarray studies are detailed in Table-2.

Example 1

SHROOM3 is Upregulated and Associated with Progression of CAN

To identify genes that could potentially contribute to the developmentof CAN we performed an interim analysis for the first 66 subjects of thecohort who had the gene expression microarray data from the 3-monthallograft biopsy as well as eGFR_12 and CADI_12. A list of candidategenes involved in CAN progression—that is, genes whose expression in the3-month allograft sample correlated with a low eGFR_12 as well as a highCADI_12—were identified and ranked. SHROOM3 was among the top-rankedgenes on the list (Table 3). Since the interim analysis, we havecollected and analyzed additional samples. At the time of preparation ofthe instant application, 3-month allograft gene expression profiles havebeen performed on 160 allografts and of which 12-month eGFR (eGFR 12)was available in 147 subjects and 12-month CADI (CADI_12) was availablein 101 subjects (FIG. 1). When we reexamined the correlation of 3-monthallograft SHROOM3 expression in the 147 subjects who had eGFR_12available, 3-month allograft SHROOM3 expression correlated inverselywith eGFR_12 (r=−0.2192, P<0.01, FIG. 2A). Of the 101 subjects who hadCADI_12, 3-month allograft SHROOM3 expression on gene expressionmicroarray analysis correlated linearly with CADI_12 (P=0.03, FIG. 2B),which remained significant (P=0.02) when 2 of the allograft samples wereexcluded from the analysis—one with BK-virus nephropathy and anotherwith cortical scarring. No correlation was identified, however, between3-month allograft SHROOM3 expression and simultaneous 3 month CADI(n=135; r=−0.1273, P=0.14, (FIG. 3D). The relationships of 3-monthSHROOM3 expression to CADI_12 and eGFR_12 were further validated byquantitative real-time polymerase chain reaction (qRTPCR) analysis in aninternal cohort of 32 subjects (r=−0.3873, P=0.02 for eGFR_12 andr=0.3774, P=0.03 for CADI_12, (FIGS. 3C & 3D). We also found a robustcorrelation between microarray and qRTPCR SHROOM3 expression (n=32,r=0.5613; p=0.0008, (FIGS. 2B and 2C). The relationship betweenLog-SHROOM3 expression and 12 m-CADI was strongest in DDRs (p<0.01)(FIG. 2D). In multivariate analysis only cold-ischemia time and presenceof acute rejection had significant effect on CADI_12. SHROOM3 expressionremained significant in deceased donors (p=0.02). Of the 160 subjectswho had 3-month allograft SHROOM3 expression examined by microarray, 85had both 3- and 12-month CADI scores available. To further corroboratethat SHROOM3 expression is associated with progression of CAN wecompared SHROOM3 expression between allografts that had ≥2 increase inCADI score (n=17; progressors) to those with less than <2 increments(n=68; non-progressors) between 3- and 12-month biopsies. SHROOM3expression was significantly higher in progressors compared tonon-progressors (p=0.04).

Example 2

Association of SHROOM3 and CAN Progression Exists in Caucasian-DonorAllografts

Since a genetic variant of SHROOM3 (SNP variant rs17319721) isassociated with CKD when studied in predominantly Caucasian cohorts[Kottgen A, 2009], we sought to determine whether the relationshipobserved between SHROOM3 and CAN followed a racial predilection. Of the147 allografts with available eGFR_12, 109 were from Caucasian donorsand their SHROOM3 expression was inversely correlated with eGFR_12(r=−0.2712, P=<0.01, FIG. 4A). Among 101 allografts with availableCADI_12, 80 were from Caucasian donors and SHROOM3 expression in thoseallografts significantly correlated with CADI_12 (P=0.02, FIG. 4B). Innon-Caucasian allografts, SHROOM3 expression was not significantlycorrelated to either eGFR_12 (n=38) or CADI_12 (n=21) (FIG. 5B, 5C).Among the 85 patients with CADI_3 and CADI_12, 70 receivedCaucasian-donor kidneys. In them, we compared Shroom3 expression betweenprogressors and non-progressors. SHROOM3 expression for the 14allografts that developed ≥2 increments in CADI was significantly higherthose with <2 change in CADI (n=56) (FIG. 3C). SHROOM3 expression in the3-month allograft biopsy was also significantly different betweenallografts with varying severity of CAN based on CADI_12: low (CADI 0-1;n=49), intermediate (CADI 2-5; n=24), high (CADI>5; n=7) (P=0.03, FIG.3C).

Example 3

A Non-Coding SHROOM3 Variant in the Donor is Associated with IncreasedSHROOM3 Expression

The A-allele (minor allele) of a non-coding SHROOM3 SNP at rs17319721has been strongly linked to chronic kidney disease by eGFR-creatinine[Kottgen A, 2009; Boger C, 2011]. We performed targeted SNP genotypingfor the rs17319721 variant in 540 allograft recipients and 468 donorsamples. Allelic prevalence of the risk allele (A) was 36.66% amongrecipients and 40.02% among donors. In both recipients and donors, weobserved that the prevalence of the risk allele was significantly higherin Caucasians compared to non-Caucasians (P<0.0001) (FIG. 6A). Since ahigher SHROOM3 expression correlates with CAN progression and the riskallele of SHROOM3 is associated with CKD in predominantly Caucasiancohorts, here we examined whether allograft SHROOM3 expressioncorrelated with the presence of the risk allele. Of the 160 allograftsfor which SHROOM3 expression was available, targeted SNP genotyping ofthe rs17319721 variant was performed in 136 cases where either donorblood samples or pre-perfusion allograft biopsy samples were available.SHROOM3 expression was significantly higher in allografts that werehomozygous for the risk allele (A/A, n=14) compared to allografts thatwere homozygous for G allele (G/G, n=52, P=0.01, FIG. 6B). SHROOM3expression of A/G-allografts (n=70) was not significantly different fromA/A or G/G. However, SHROOM3 expression was significantly higher inallografts from donors with at least one risk allele (A/A or A/G; n=84)compared to donors without the risk allele (G/G; n=52, P=0.02). Whenspecifically examined in 103 Caucasian-donor allografts and 33non-Caucasian-donor allografts the relationship between SHROOM3expression and the risk allele in the allograft remained constant butdid not attain statistical significance (P=0.05 in Caucasian-donorsFIGS. 7A & 7B respectively). Interestingly, when we examined therelationship of SHROOM3 expression to recipient-genotype rather than thedonors, there was no significant correlation between risk allele andexpression in 147 recipients (FIG. 7C).

Example 4

Risk Allele of SHROOM3 is Associated with Diabetic Mellitus as the Causeof ESRD and CKD in Caucasians

Since the risk variant of SHROOM3 has been linked to CKD, we examinedwhether the risk allele is associated with a particular etiology ESRD inour cohort of recipients. As the risk allele of SHROOM3 was differentlydistributed between the different ethnicities (Tables 4a & 4b), we haverestricted all subsequent comparisons between donors and recipients tothe same ethnicity. We observed that the allelic frequency of the riskallele was not significantly different between Caucasian donors andrecipients (42.87% vs. 43.07% respectively). As only 114 of the 468donors were non-Caucasians, the number of subjects in each non-Caucasianethnicity (i.e. AAs, Asians, Hispanics and others) was insufficient tomake valid inference about SHROOM3 genotype and its relationship to ESRDetiology. When we analyzed the allele prevalence among Caucasianrecipients according to their documented ESRD diagnosis (excludingrecipients who had previous transplants, congenital diseases, or unknownetiology of ESRD), we noted that the risk allele was most prevalent inCaucasian recipients with diabetes mellitus as their primary ESRDdiagnosis (47.25%; n=126). We found that Caucasian recipients withdiabetes alone without hypertension had an unadjusted odds ratio of1.418 (95% CI=1.000-2.11; p=0.04) while patients with diabetes withhypertension had an odds ratio 1.36 (95% CI=1.031-1.814; p=0.029) ofhaving the risk allele when both were compared to all Caucasian donorswho were not related to the recipients (FIG. 8A). Furthermore, theallelic prevalence for the risk allele in patients with diagnosis otherthan diabetes and HTN as causes for ESRD was 38.36% (n=159), which wasnot significantly different from allelic frequency in unrelatedallograft donors (39.64%). For external validation of the association ofthe risk allele with diabetic kidney disease, we analyzed an independentcohort of subjects. Among the Caucasian participants of the study(n=3782), 763 were identified as having CKD. Again allele prevalence wassignificantly higher in those with Diabetes and CKD (n=138) compared tothose without CKD (n=3019) and, those without CKD or Diabetes (n=2691)(50.4% vs. 40.4% vs 39.3%, P<0.01) (FIG. 8B). In multivariate analysiswithin the Caucasian cohort using age, BMI, hypertension, diabetes,family history of diabetes or kidney disease as covariates, adjustmentfor diabetes negated any effect of the risk allele on CKD as an outcome.The allele prevalence was also similar in non-diabetics with and withoutCKD (38.6% vs 39.26% respectively). Within the CKD cohort, AAs with CKD(n=721) and, Diabetes with CKD (n=204) had similar allele distributionas those without CKD (n=2729) (20.59% vs 18.46% vs 21.78%; P=0.8159)suggesting that the SHROOM3 SNP does not play a significant role indiabetic/non-diabetic kidney disease in this group.

Example 5

Risk-allele of rs17319721 enhances SHROOM3 expression throughTCF4-mediated transcriptional activation rs17319721 is located withinthe first intron of SHROOM3. Since the A allele is associated with ahigher expression of SHROOM3, we sought to understand the effect of theG-to-A substitution on the transcriptional regulation of SHROOM3. Whenwe examined the intronic region of SHROOM3 containing rs17319721, wefound that the G-to-A substitution generates a potential consensusbinding sequence for transcription factor 4 (TCF4/TCF7L2), a highmobility group (HMG) box-containing transcription with the consensusbinding sequence of 5′-(A/T) (A/T)CAAAG-3′. TCF4 is involved in theWnt/β-catenin signaling pathway [Wortman B, 2002; Henderson L J, 2012].Additionally in our microarray analysis, patients in the highestquartile of Shroom3 expression also had significantly upregulated TCF7L2and Beta-Catenin (P<0.0001). To further examine whether this intronicregion containing the rs17319721 SNP possesses any function as anenhancer of SHROOM3 transcription, we generated two SHROOM3promoter-enhancer luciferase reporter constructs that consisted of a 3Kb SHROOM3 promoter region and a 100 bp sequence from the first intronof SHROOM3 containing either the A-allele or the G-allele of thers17319721 (FIG. 9. Construct maps). The SHROOM3 reporter construct withthe A-allele had a higher increase in luciferase-to-renilla reporteractivity compared to the G-allele without TGFβ stimulation and with TGFβstimulation there was an increase in activity. Treatment with either aTCF4 inhibitor, BC21, or a β-catenin inhibitor Quercetin, abrogated thedifference in reporter activity between the A- and G-allele reporterconstructs (FIG. 10A) which further confirmed that TCF4-enhancersequence is responsible for the difference in the expression of the A-vs G-allele reporter constructs. To further confirm TCF4 binding to the100-bp SHROOM3 intronic region we performed EMSA. TCF4 binding to theintronic sequence containing the A-allele was more than the G-allele,which was abrogated in samples with excess cold oligos (FIG. 10B)

Example 6

TGFβ1 Enhances SHROOM3 Expression in a β-Catenin/TCF4-Dependent Manner

Since SHROOM3 is regulated by TGFβ1 in HK-2 cells [Brennan E P, 2012]and TGFβ1 is a key growth factor mediating renal injury and fibrosis[lan H Y 2012] we further characterized TGFβ1-mediated regulation ofSHROOM3. We found that TGFβ1 treatment of PRCEC increased SHROOM3 mRNAexpression maximally at 5 ng/ml (FIG. 11A) and protein expression at 48hours (FIG. 11B). Analysis of the SHROOM3 promoter sequence—up to −10 kbfrom the transcriptional start site—using a transcription factor bindingmotif prediction program (TRANSFAC) did not reveal any Smad-bindingsequence, suggesting that the TGFβ1-induced increase in SHROOM3expression is not due to canonical TGFβ1/SMAD signaling. As TGFβ1 isknown to crosstalk with the Wnt/β-catenin/TCF4 pathway and TCF4regulates SHROOM3 expression, we tested whether TGFβ1-induced SHROOM3expression is dependent on β-catenin/TCF4 interaction. We found thatboth BC-21 and quercetin abrogated the TGFβ1-induced increase in theexpression of SHROOM3 protein (FIG. 12A) and mRNA (FIG. 12B), thusconfirming that TGFβ1-induced SHROOM3 expression is β-catenin/TCF-4dependent.

Example 7

SHROOM3 Facilitates Canonical TGFβ1/SMAD3 Signaling and Profibrotic GeneExpression

We investigated whether SHROOM3 has any impact on TGFβ1-mediatedpro-fibrotic gene program, which is a well-characterized driver ofkidney fibrosis in CKD as well as CAN [Lan H Y 2012; Campistol J M2001]. First we compared the expression of TGFβ1-target genes related totissue fibrosis in PRCEC with or without SHROOM3 overexpression thatwere treated with either TGFβ1 or vehicle. Overexpression of SHROOM3 wasconfirmed by RTPCR (FIG. 13A) and Western blot (FIG. 13B). Theexpression of profibrotic TGFβ1-target genes, including COL1A1 and FN1,were increased by TGFβ1 treatment alone as well as SHROOM3overexpression alone (FIG. 14A). TGFβ1-induced expression of COL1A1 wasfurther increased in cells with SHROOM3 overexpression compared to thosewithout SHROOM3 overexpression (Vector+TGF). To further characterize howSHROOM3 facilitated TGFβ1 signaling we investigated the phosphorylationof SMAD-3 which indicates activation of canonical TGFβ1/SMAD signalingin PRCEC. Cells with or without SHROOM3 overexpression were treated witheither TGFβ1 or vehicle. Phosphorylation of Smad3 in TGFβ1-treated cellswas enhanced by SHROOM3 overexpression compared to vector-transfectedcells (FIG. 14B). TGFβ1 treatment of SHROOM3-overexpressed PRCEC alsodeveloped more prominent F-actin bundles compared to TGFβ1-treated cellswithout SHROOM3 overexpression (FIG. 14C-Upper and lower Panels). Next,we sought to determine whether TGFβ1-induced profibrotic gene program isdependent on SHROOM3. SHROOM3 knockdown in PRCEC significantly reducedCOL1A1 and FN1 transcripts (FIG. 15A). TGFβ1-induced expression ofCOL1A1 was also significantly attenuated in SHROOM3 knockdown cellscompared to cells transduced with the empty lentivector. TGFβ1-inducedexpression of FN1, however, was not affected by SHROOM3 knockdown.Phosphorylation of SMAD3 in SHROOM3-knockdown cells was significantlyreduced at 30 min, but not at 15 min, after TGFβ1 stimulation comparedto cells without SHROOM3 knockdown (FIG. 15B). When taken together theseresults suggest that SHROOM3 facilitates TGFβ1/SMAD3-inducedpro-fibrotic gene expression program. Further supportive of this was ourfinding in the microarray cohort of CTGF, Vimentin, Collagen-IV(downstream of TGF/SMAD3 signaling) were among genes significantlyupregulated in patients in the highest quartile of SHROOM3 expression.

Example 8

A-Allele of rs17319721 in the Donor is Associated with Higher AllograftSHROOM3 Expression at 3 Months.

Multiple studies have now linked the rs17319721 SHROOM3 SNP to CKD20-22.Whether the risk allele is associated with altered SHROOM3 expression inthe renal parenchyma, or is related to CAN is not known. As of Jan. 1,2013, five hundred eighty nine recipients have been enrolled in theparent study. We performed targeted genotyping for this locus on 540allograft recipients and 517 donors within our cohort. Allelicprevalence of the CKD-associated A-allele was 36.66% among recipientsand 39.94% among donors. Overall, the prevalence of the A-allele wassimilar for Caucasian donors and recipients (42.87% vs. 42.56%respectively). The number of subjects in each non-Caucasian ethnicity(i.e. AAs, Asians, Hispanics and others) was not sufficient to makevalid inference about rs17319721 distribution. In both recipients anddonors, we observed that the prevalence of the A-allele is significantlyhigher in Caucasians compared to non-Caucasians (P<0.0001).

Next, we examined whether allograft SHROOM3 expression at 3-months(SHROOM3-3M) correlated with the presence of the A-allele. Allograftgene expression microarray analysis from 3-month protocol biopsies wasperformed on 159 out of the entire 589 enrollees in this study. Thesepatients represent by chronology the first 159 enrollees who werebiopsied 3 months after transplantation. Both targeted genotypingresults for rs17319721 and SHROOM3-transcript levels from kidneyallografts were available from 136 donors and 145 recipients. Weobserved that SHROOM3-3M was significantly higher in allografts thatwere homozygous for the CKD risk allele (A/A, n=14) compared toallografts that were homozygous for the G allele (G/G, n=52, P=0.01).SHROOM3-3M was also significantly higher in allografts from donors withat least one risk allele (A/A or A/G; n=84) compared to donors withoutthe risk allele (G/G; n=52, P=0.02). Interestingly, when we examined therelationship of SHROOM3-3M with respect to the recipient's genotype,rather than the donor's, there was no significant correlation betweenthe A-allele and SHROOM3-3M (n=145).

Example 9

Allograft SHROOM3 Expression at 3-Months and A-Allele of rs17319721 areAssociated with Higher Risk of CAN in Renal Allograft Recipients.

Since we observed that the A-allele of rs17319721 is associated withSHROOM3 transcriptional activation and, that increasedSHROOM3-expression facilitated TGF-β1 signaling in PRCEC, we examinedwhether SHROOM3-3M and/or the donor risk-genotype correlated withindices of allograft dysfunction (CAN) at 12-months.

Allograft gene expression microarray analysis from 3-month protocolbiopsies was performed on 159 out of the entire 589 enrollees in thisstudy. At the time of this filing, eGFR-12 was available in 147 subjectsand CADI-12 was available in 101 subjects from the subgroup. Reasons fornot having a 12-month biopsy in this subgroup included graft loss (n=8),death (n=1), lost-to-follow up (n=9), contraindication for or inabilityto obtain a renal allograft biopsy (n=40).

SHROOM3-3M correlated inversely with eGFR-12 (r=−0.2192, P<0.01) andpositively with CADI-12 (r=0.2458, P=0.03). This correlation remainedsignificant (P=0.01) after exclusion of 2 biopsies with diagnosis(BK-virus nephropathy and severe cortical scarring). The relationshipbetween SHROOM3-3M and CADI-12 was stronger in deceased-donor allografts(p<0.01). The relationships of SHROOM3-3M to CADI-12 and eGFR-12 werefurther validated by qRTPCR in an internal cohort of 32 subjects(r=−0.39, P=0.02 for eGFR-12 and r=0.38, P=0.03 for CADI-12). A robustcorrelation existed between SHROOM3 expressions from microarray andqRTPCR (P=0.0008). No correlation existed, however, between SHROOM3-3Mand simultaneous 3-month CADI (n=135). SHROOM3-3M was predictive ofCADI-12 greater than 2 (CADI-12≥2) and inversely related to eGFR-12 inmultivariate analysis. Among covariates included in the analysis, acuterejection before 3 months had significant independent effects onCADI-12, and on eGFR-12 (P<0.05).

To corroborate that SHROOM3-3M is associated with progression of CAN wecompared SHROOM3-3M between allografts that had ≥2 increase in CADIscore (ΔCADI≥2, n=17, known as Progressors) to those with less than <2change in CADI score (ΔCADI<2, n=68, known as Non-progressors) between3- and 12-month biopsies. To minimize the effect of baseline disease onsubsequent histological progression, we excluded allografts withCADI-3>2 from this analysis. SHROOM3-3M was significantly higher in theProgressors compared to the Non-progressors (P=0.04).

Next we examined the donor risk genotype and its association with CAN.At the time of analysis, two-hundred and three subjects of the cohorthave had CADI scores reported at 12 months—101 from the microarraycohort and 102 from the non-microarray cohort. In this group, thepresence of the A-allele in the donor was associated with asignificantly greater risk of a CADI-12≥2 in all allografts (OR=1.98,CI=1.10-3.59), indicating a higher risk of CAN with the risk allele.

Example 10

SHROOM3 Facilitates Canonical TGF-β1/SMAD3 Signaling and ProfibroticGene Expression in a Murine Model In Vivo:

Methods

To examine the mechanism of facilitation of fibrosis by SHROOM3, wedeveloped a murine model of inducible shRNA-mediated SHROOM3 knockdown.In our model, reverse tetracycline-controlled transactivator(RTTA)-elements were linked to the universal ROSAm26 promoter for RTTAexpression in all cell-types. After in vitro validation, twoSHROOM3-specific shRNA hairpins were linked todoxycycline-RTTA-responsive elements and positioned 3′ to the Collagen-1gene. Sample genotyping PCR for Rosa-RTTA element and shRNA (COLTGM)sequences and southern blot gel electrophoresis are displayed (FIG.16A). Doxycycline (DOX) feeding to induce Shroom3 knockdown in genotypedmice was initiated 3-weeks prior to UUO surgery and continued until thedate of sacrifice 10-days later. To study the development of renalinterstitial fibrosis, we performed unilateral ureteric obstructionsurgery (UUO) on 8-10 week old animals after 3-weeks of DOX-feeding (n=5in each shRNA clone). Mice were sacrificed at 10-days post UUO.SHROOM3-shRNA animals of the same age that were not fed with DOX wereused as controls. Results were analyzed quantitatively by the unpairedt-test.

Results

We examined the impact of shRNA-mediated SHROOM3 knockdown onTGF-β1/SMAD3 signaling and renal fibrosis in the above DOX-inducibleshRNA mouse strain. Doxycycline (DOX) feeding of these animals confirmedSHROOM3 knockdown (˜75%) by 3-weeks (real-time PCR (RT-PCR) and Westernblot (WB) from renal cortical lysates (FIGS. 16B and 16D). DOX-fedanimals showed significantly inhibited phosphorylation of SMAD3 in UUOkidneys by Western blot (FIG. 16B). COL1A1 production in UUO kidneys wasinhibited with Shroom3 knockdown as shown by RTPCR of kidney lysates andby immunofluorescence (IF) in tissue sections (FIG. 16C—lower panel andgraph; and 16D). Renal interstitial fibrosis measured by picrosirius redstaining (FIG. 16C—middle panel), was significantly abrogated in Shroom3knockdown animals. These results validate the role of SHROOM3 incanonical TGF-β1 signaling.

Discussion

Chronic allograft nephropathy remains a substantial cause for allograftfailure and RRT {Paul L C, 1999} {Chapman J R, 2005} {Racusen L C, 2010}{USRDS 2012}. Modern immunosuppressive strategies have had significantimpact on short term allograft outcomes with much less improvement inlong-term outcomes {Hariharan S, 2000} {Meier-Kriesche H U, 2004} {LambK E, 2011}. Further, CAN remains a histological entity with arbitrarystages and variability between reporting pathologists {Solez K, 2008}{Akalin E, 2010} {Ying L, 2009} {Halloran P, 2002}. Therefore,identification of newer markers for potential early diagnosis andtherapy of CAN is imperative. In the GoCAR study, we are examining theability of allograft gene-expression profiles from protocol biopsies at3-months to predict the development of CAN and TIF at 12 months. We thusidentified Shroom3 as a novel candidate gene whose allograft expressionprecedes and predicts the derangement of renal function and theprogression of TIF.

While evolved CAN has shown distinct transcriptional signatures in priorstudies {Flechner S, 2004} {Donauer J, 2003}, issues have been raisedregarding their interpretation and generalizability. Development ofbiomarkers and/or therapeutic strategies has been impeded by largegene-panel sizes, small sample sizes, single time point biopsies,heterogeneity of gene chip assay used and low fidelity of pre-arrayamplification techniques {Ying L, 2009} {Akalin E, 2010}. Studies basedon for-cause biopsy transcriptional profiles are less reliable fordeveloping predictive panels or therapeutics for CAN due to genediversity dependent on pathology at the time of biopsy. To determine agene-signature that would predict the development of CAN, Scherer et al,profiled amplified RNA (Affy HG-U95Av2 chip) from 6-month protocolbiopsies of 17 patients, 12 of whom went on to develop CAN. Theydeveloped a 10-gene cluster that was 88% predictive of developingchronic rejection at 12-months. The relationship however, was notsignificant when single genes were analyzed {Scherer A, 2003}. We usedunamplified RNA from 3-month biopsies and whole-exon gene chip array (˜4probes/exon, ˜40 probes/gene) to correlate differentially expressedgenes with eGFR and CAN at 12 months in our larger cohort of patients.Furthermore, Shroom3 expression in our study retained its significancewhen analyzed alone in the entire cohort with better correlation in thesubset of DDRs and WDKRs. This was validated in our smallerinternal-external cohort of patients by RT-PCR. Importantly, meanShroom3 transcript levels were significantly higher in patients whoseCADI-scores progressed (Delta CADI≥2) between 3 and 12 months comparedto those with relatively stable histological scores. This implies a rolefor Shroom3 in the progression of fibrosis and CAN.

TIF is a common histological end-point for CAN and CKD. Consequently,genes linked to fibrogenesis and EC-matrix production, specificallyrelated to TGF-beta signaling, have emerged as differentially regulatedfrom transcriptional studies in patients with CAN and animal models ofCKD {Flechner S, 2004} {Hotchkiss H, 2006} {Mas V, 2007} {Ju W, 2009}. ASNP in Shroom3 has emerged independently linked to incident andprevalent CKD in Caucasian predominant cohorts {Kottgen A, 2009} {BogerC, 2011}. However, Shroom3 gene function and its relationship to loss ofeGFR and TIF are hitherto unknown. In PRCEC, our RT-PCR studies suggesta small but significant overproduction of EC-matrix markers(Collagen-land Fibronectin) by Shroom3 overexpression and a markedsuppression of these markers with SiRNA mediated Shroom3 inhibition.Further, Shroom3 overexpression appears to facilitate canonical TGF-betasignaling as evidenced by enhanced Smad-3 phosphorylation in PRCEC.Consistent with this is the amplified response of profibrotic markerproduction (SNAIL, MMP-2, collagen-1) upon TGF-beta treatment inShroom3-transfected cells compared to controls. Also in line with theseobservations are the suppressed P-Smad3 and Collagen-1 levels weobserved with Shroom3 SiRNA in TGF-treated cells. TGF-beta alsoincreased Shroom3 expression in PRCEC. Other groups have made thisobservation in HK-2 cells using RNA-sequencing {Brennan E P}. This alongwith the salutary effect of increased Shroom3 on TGF-beta signaling mayindicate a positive feedback loop between Shroom3 and TGF. Together,these suggest that higher Shroom3 expression may have profibroticeffects in renal epithelial cells. Interestingly, from our biopsy data,having one or two copies of the risk-allele (A) in the donor appears tosignificantly increase Shroom3 transcript levels in biopsy tissue (˜1.4fold). There was no consistent association between recipient-SNP andShroom3 expression suggesting little contribution of allograft Shroom3expression from infiltrating recipient cells. In summary, this impliesthat the association between the SNP and CKD in prior GWAS studies maybe explained by increased kidney Shroom3 levels and a subsequentprofibrotic response that go along with having the risk allele.

In univariate analysis the association between Shroom3 expression andeGFR/CADI was significant in WDKRS and DDRs but not in non-WDKRs orLDRs. Notably, of the DDRs, 51/59 were WDKRS. The effect—alleleprevalence was also significantly higher in whites compared tonon-whites in our analysis. Prior GWAS studies that identified Shroom3involved Caucasian predominant cohorts {Kottgen A, 2009} {Boger C,2011}. In a study to identify susceptibility loci for Urinary-albumincreatinine ratio (UACR), the Shroom3 SNP retained association to eGFRand UACR in whites but did not attain significance in African-Americans{Ellis J W, 2011}. Our observation of the lack of association betweenShroom3 expression and eGFR/CADI in non-whites is similar to thesepublished results. The increased Shroom3 expression with the presence ofthe A-allele in the donor, however, was also observed in non-WDKRs.Further the mean Shroom3 expression by microarray was not significantlydifferent between WDKRs and non-WDKRS (data not shown). Hence, theinsignificant effect of Shroom3 on eGFR/CADI in non-whites is unclear.Non-WDKRs in the microarray (n=21) and RT-PCR (n=5) were fewer thanWDKRs. More conjecturally, a polymorphism having greater impact onallograft outcomes in non-WDKRs that we did not analyze may bedifferently distributed within this cohort. In the cohorts reported onherein, European-Americans with diabetic ESRD and CKD respectively hadthe highest prevalence of the risk allele of rs17319721. However, theFamily Investigation of Nephropathy and Diabetes (FIND) study did notreport linkage between loci on chromosome 4 and diabetic nephropathy inCaucasians {Igo R P, 2011}. The inconsistency between these results maystem from our comparison group which included only kidney donors whowere not related.

All patients in the 3-month microarray did not have biopsies for outcomeassessment at 12-months (17 allograft losses, 42 lost follow-up orrefused 12-month biopsy). The sample size of 101 patients is stillrobust in comparison to prior transcriptional studies in allograftrecipients {Flechner S, 2004} {Donauer J, 2003} {Sarwal M, 2003} {ReeveJ, 2009}. Only 36 patients had sufficient quality RNA for RT-PCRvalidation after microarray—though the relationships were significantwithin this sample. While the facilitation of TGF-beta signaling byShroom3 excess was observed in vitro, the mechanism of this interactionis uncertain. The C-terminal ASD-2 domain of Shroom3 has been shown tobe essential for Rho-Kinases 1&2 (ROCKs) recruitment and function in theinvaginating neural tube. Mutation of this domain leads to loss ofROCK-function {Nishimura, 2008}. In chondrocytes, ROCKs facilitated andROCK-inhibitor (Y27632) inhibited Smad3-phosphorylation with TGF-betatreatment {Xu T, 2012}. Further, in animal models of unilateral ureteralobstruction, ROCK-inhibitors retarded renal interstitial fibrosis andGFR-decline {Satoh S, 2002} {Nagatoya K, 2002} {Takeda Y, 2010}. Thoughwe did not test this, the mechanism of interaction with TGF-beta may inpart be through Shroom3-mediated ROCK facilitation. Finally, thers17319721 locus on the Shroom3 gene is intronic, between exons 1 and 2.The reason for the apparent regulatory effects of this region is lessclear from our observations. Recently, the results from the Encyclopediaof DNA elements (ENCODE) consortium have been sequentially publisheddescribing the regulatory functions attached to intronic loci within thehuman genome {Dunham I, 2012} {Neph S, 2012} {Gerstein M, 2012}. Studiesusing histone methylation mapping suggest that the Shroom3 SNP islocated in an area predicted to have enhancer function {Dr KatalinaSusztak, ASN 2012}. This is consistent with our findings.

In summary, Shroom3 is a novel candidate gene whose expression in therenal allograft precedes and predicts the derangement of renal functionand TIF in CAN. Higher allograft Shroom3 levels appear to predicthistological progression of CAN. We also show that these relationshipsare best in recipients of white-donor kidneys and deceased-donorkidneys. Our findings confirm for the first time that the previouslydescribed CKD-associated Shroom3 locus (rs17319721) {Kottgen A, 2009}{Boger C, 2011} mediates its effect through increased Shroom3expression. Finally our in vitro studies suggest a salutary role forShroom3 in canonical TGF-beta signaling and type I collagen productionpromise as a therapeutic target in both CAN and CKD to reduce theprogression of TIF and retard ESRD.

The present invention also includes a kit for use in identifyingpatients suffering from kidney diseases and for predicting theprogression of kidney fibrosis and TIF in renal allograft recipients.The kit consists of reagents for RT-PCT analysis of Shroom3 expressionand a microassay for detecting the rs 17319721 SNP risk allele, apositive control for the RT-PCR assays, buffers and instructions foruse. The kit is used by obtaining a renal biopsy from a patient whoreceived a kidney transplant from a donor, determining Shroom3expression and comparing a level of Shroom3 expression in biopsyspecimen with the level of Shroom expression in the positive control inthe kit, all by RT-PCR. The microarray is used to identify kidney donorswho are homozygous for the Shroom3 A allele.

TABLE 1 Demographics of GOCAR enrollees Mean ± SD Demographics N (%)[range] Recipient Age: All Recipients 589 50.17 yrs [18-83] RecipientGender (Percent females) 185 (31.41) Recipient Race: White (W) 375(63.37) African-American (AA) 123 (20.88) Asian (A)  34 (5.77) Hispanic(H)  34 (5.77) Other (O)  16 (2.71) Recipient ESRD diagnosis- Allrecipients: Diabetes only 115 (19.42) Hypertension only  97 (15.28)Diabetes & Hypertension  90 (15.28) Polycystic disease  53 (8.99)Glomerular disease (including 107 (18.17) FSGS/IgA) Unknown  16 (2.72)Prior transplants  13 (2.38) Others  98 (16.64) Donor Age 589 42.01 yrs[0-76] Donor Gender (Percent females) 278 (47.20) Donor Race: White (W)451 (76.57) African-American (AA)  57 (9.68) Asian (A)  17 (2.89)Hispanic (H)  45 (7.64) Other (O)  19 (3.23) Donor type: Deceased-Donors(DD) 329 (55.86) Living-Related Donors (LRD) 147 (24.96)Living-Unrelated Donors (LURD) 113 (19.19) Donor SNP analysis 468Recipient SNP analysis 540

TABLE 2 Demographics of patients in the microarray studies (CADI & eGFRanalysis) Mean ± SD Demographics N (%) [range] Recipient Age: 160 (100)48.84 ± 13.27 [19-73] Recipient Gender (Percent  47 (29.35) females)Recipient Race: White (W)  92 (57.5) African-American (AA)  37 (23.1)Asian (A)  10 (6.25) Hispanic (H)  14 (8.75) Other (O)  7 (4.38) DonorAge 160 41.13 ± 16.80 yrs [3-76] Donor Gender  77 (48.13) (Percentfemales) Donor Race: White (W) 121 (75.63) African-American (AA)  17(10.63) Asian (A)  7 (4.38) Hispanic (H)  12 (7.5) Other (O)  3 (1.88)Donor type: Deceased-Donors (DD)  95 (59.38) Living-Related Donors (LRD) 35 (21.88) Living-Unrelated Donors  30 (18.75) (LURD) CADI analysis(3-months) 135  1.18 ± 1.76 [0-9] CADI analysis (12-months) 101  2.09 ±2.49 [0-10] eGFR analysis (6-months) 139 57.40 ± 17.4 ml/min[15.08-116.37] eGFR analysis (12-months) 147 58.12 ± 19.45 ml/min[10.66-109.24]

TABLE 3a SNP distribution in genotyped Donors and Recipients in theGoCAR cohort (Donors = 468, Recipients = 540) Whites Non-WhitesGenotypes Donor Recipient Donor Recipient A/A  57 (16.1)  59 (16.7)  13(11.4)  10 (5.4) A/G 190 (53.7) 187 (52.8)  44 (38.6)  65 (34.9) G/G 107(30.2) 108 (30.50)  57 (50) 109 (58.7) A-allele (%)  42.86  43.07  41.67 30.56 Total 354 354 114 186

TABLE 3B Demographics & Race-wise distribution of non-white donors (114)and non- white recipients (186) Afro-American Hispanic Asian Other*Genotypes Donor Recipient Donor Recipient Donor Recipient DonorRecipient A/A 1 (1.8) 4 (3.6) 9 (21.9) 6 (19.4) 1 (7.1) 0 (0) 2 (33.3) 2(11.1) A/G 23 (43.4) 43 (39.1) 17 (41.5) 12 (38.7) 3 (21.4) 3 (11.1) 1(16.6) 7 (38.9) G/G 29 (54.8) 63 (57.3) 15 (36.6) 13 (41.9) 10 (71.4) 24(88.9) 3 (50) 9 (50) A-allele (%) 23.58 23.18 42.68 38.70 17.85 11.1141.67 30.56 Total 53 110 41 31 14 27 6 18

TABLE 4 QPCr primer sequences Forward primer Reverse primer Gene name(5′ → 3′) (5′ → 3′) HGAPDH TGTTGCCATCAATGA CTCCACGACGTACTC CCCCTT AGCG(SEQ ID NO: 1) (SEQ ID NO: 2) Shroom3 CCCTCTCGGGGCGTC GCCCAGCACTACTCGTAGCC CTCC (SEQ ID NO: 3) (SEQ ID NO: 4) Collagen-1 GATGGTGAAGATGGTGCCCAAGTCCAACTC CCCAC CTTTT (SEQ ID NO: 5) (SEQ ID NO: 6) Fibronecctin-1TCCAGGAGTTCACTG CTGCAAGCCTTCAAT TGCC AGTCA (SEQ ID NO: 7) (SEQ ID NO: 8)SNAIL ACCACTATGCCGCGC GGTCGTAGGGCTGCT TCTT GGAA (SEQ ID NO: 9)(SEQ ID NO: 10) Matrix ACCCAGATGTGGCCA GAGCAAAAGGCATCA Metallo- ACTACTCCACT proteinase-2 (SEQ ID NO: 11) (SEQ ID NO: 12) VimentinTTGACCTTGAACGCA GCTGTTCCTGAATCT AAGTG GAGCC (SEQ ID NO: 13)(SEQ ID NO: 14) E-Cadherin CAGCACGTACACAGC ACCTGAGGCTTTGGA CCTAA TTCCT(SEQ ID NO: 15) (SEQ ID NO: 16) Slug GATGCATATTCGGAC CCTCATGTTTGTGCACCACAC GGAGAG (SEQ ID NO: 17) (SEQ ID NO: 18) FSP-1 GCCCTGGATGTGATGTCGTTGTCCCTGTTG GTGT CTGTC (SEQ ID NO: 19) (SEQ ID NO: 20) SMACAACCGGGAGAAAAT TAGATGGGGACATTG GACTC TGGGT (SEQ ID NO: 21)(SEQ ID NO: 22)

TABLE 5 Primer sequences designed for RT-PCR andgenerating Luciferase-Reporter plasmid constructs Forward primerReverse primer Primers (5′ → 3′) (5′ → 3′) HGAPDH TGTTGCCATCAATGACTCCACGACGTACTC CCCCTT AGCG (SEQ ID NO: 23) (SEQ ID NO: 24) SHROOM3CCCTCTCGGGGCGTC GCCCAGCACTACTCG TAGCC CTCCC (SEQ ID NO: 25)(SEQ ID NO: 26) Constructs TTATAGGTACCTTGA TTAAGCTTCCATGCC Wild-typeGACAATAGAGTTGCC AAACACATGATCCCT (promoter (SEQ ID NO: 27) C only)(SEQ ID NO: 28) A-allele TTTGGTACCGAGTAG TTAAGCTTCCATGCC ConstructCAGGGCAAAAACAAA AAACACATGATCCCT AGCCCTTGAGACAAT C AGAGTTGCC(SEQ ID NO: 30) (SEQ ID NO: 29) G-Allele TTTGGTACCGAGTAG TTAAGCTTCCATGCCConstruct CAGGGCAAAAACAAA AAACACATGATCCCT GGCCCTTGAGACAAT C AGAGTTGCC(SEQ ID NO: 32) (SEQ ID NO: 31) Legend: Kpnl was introduced in allforward primer and Hind III in the Reverse ones.

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The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all values are approximate, and areprovided for description. Patents, patent applications, publications,product descriptions, and protocols are cited throughout thisapplication, the disclosures of which are incorporated herein byreference in their entireties for all purposes.

1.-17. (canceled)
 18. A method for determining if a Caucasian allograftrecipient that received a kidney allograft from a Caucasian kidney donoris at risk for allograft rejection which comprises the steps ofdetermining the levels of SHROOM 3 expression in a kidney biopsyspecimen obtained from the allograft recipient at a predetermined timeafter transplant; comparing the level of SHROOM 3 expression in thebiopsy specimen with the levels of SHROOM 3 expression in controlbiopsies obtained from normal subjects; wherein if the level of SHROOM 3expression in the allograft is higher than the expression levels ofSHROOM 3 in a control the allograft recipient is at risk for allograftrejection.
 19. The method of claim 18 comprising administering animmunosuppressive agent to the allograft recipient at risk for allograftrejection.
 20. The method of claim 19 wherein said immunosuppressiveagent is cyclosporin.
 21. The method of claim 18 which comprisesadministering an angiotensin converting enzyme inhibitor to saidallograft recipient.
 22. The method of claim 18 wherein said levels ofShroom3 expression are determined by Real Time Polymerase Chain Reaction(RT-PCR).
 23. The method of claim 18 wherein said allograft recipient atrisk for allograft rejection is suffering from a disease selected fromthe group consisting of Chronic Allograft Nephropathy (CAN), ChronicKidney Disease (CKD), obstructive uropathy, HIV-associated nephropathy,diabetic nephropathy and fibrosis.
 24. A kit for identifying renalallograft recipients at risk for developing CAN or CKD selected from thegroup consisting of obstructive uropathy, HIV-associated nephropathy anddiabetic nephropathy comprising in separate containers primers forRT-PCR SHROOM3 expression assays, a microarray for gene expressionanalysis and primers for RT-PCR analysis of the rs17319721 risk allele,buffers and instructions for use.
 25. A method for identifying the riskof developing fibrosis in a Caucasian kidney allograft recipient afterreceiving a kidney transplant from a Caucasian donor, comprising thesteps of: determining if said donor expresses the rs 17319721 SNP riskallele; conducting a genetic analysis to determine if said donor ishomozygous for said risk allele; wherein if said donor is homozygous forsaid risk allele then said allograft recipient is at risk for fibrosis.26. The method of claim 18 which comprises treating the allograftrecipient determined to be at risk for allograft rejection with ananti-rejection agent.